To characterize the biophysical properties of an artificial vitreous body substitute (VBS), which consists of a biocompatible, cross-linked, hyaluronic acid (HA)-based hydrogel, by analysing the VBS’s influence on intraocular pressure (IOP) and retinal integrity in distinct ex vivo eye models in order to evaluate the its potential for in vivo biocompatibility testing.Pig eyes were obtained immediately postmortem, and VBS was injected after core-vitrectomy. IOP was followed for 24 h (n = 5). VBS influence on retinal integrity was investigated using isolated bovine retinas superfused with an oxygen saturated nutrient solution. An electroretinogram (ERG) was recorded on explanted bovine retinae using silver/silver chloride electrodes; after application of VBS for 2 min, a washout period of 70 min was employed. The percentage of a-and b-wave reduction at the end of the washout phase was compared to baseline values (n = 5). Data were calculated throughout as the mean and the standard deviation. qRT-PCR  or western blot analysis was used to test for toxicity of Princess Volume after 24 h (and β-3 tubulin with GAPDH as a control gene). Significance was estimated by Student´s t-test; p ≤0.05 was considered to be statistically significant.The IOP increased non-significantly by 10% after 24 h. Short-term biocompatibility testing using isolated superfused bovine retinas showed neither significant reductions of the b-wave nor the a-wave amplitudes . qRT-PCR and western blot analysis did not reveal significant toxicity after 24 h.The manufactured HA-based hydrogel showed highly favourable biophysical characteristics in the explored ex vivo models, justifying in vivo studies enabling the assessment of biocompatibility. Vitreous tamponades are used for the treatment of complicated cases of retinal detachment. The most common tamponades include expansive gases and silicone oil, but their physicochemical characteristics and associated side effects limit their clinical application . Gas hasAn ideal vitreous tamponade should be injectable, transparent, biocompatible, stable for a certain period of time and fully biodegradable. It should be hydrophilic with a comparable refractive index (close to 1.33) but a higher viscosity and elasticity similar to that of the human vitreous body . This prN-acetyl-D-glucosamine connected through alternating ß-1,4 and ß-1,3 glycosidic bonds. The axial hydrogen atoms form a nonpolar, hydrophilic face, thereby creating a twisting ribbon structure. In physiological solutions, a hyaluronan molecule imitates the form of an expanded random coil structure that occupies a very large domain; therefore, HA forms a viscous compound [HA is a glycosaminoglycan co-polymer of D-glucuronic acid and compound , 6.Polymers, including HA, have been heavily investigated in the last several decades, but to date, none has fully met expectations either due to a short intravitreal half-life, inflammatory or even toxic effects at the retina and in the vitreous cavity, or little efficacy in the treatment of retinal detachments , 7.et al. with sodium hyaluronate (HYVISC) resulted in a reattached retina in only 18% of the treated patients compared to 70–90% treated with silicone oil or gas tamponades. The short residence time of less than 14 days of the hydrogel in the vitreous cavity was thought to account for the unsatisfying results. In addition, an increase in IOP was observed as an unwanted side effect [Thus, early clinical studies by Pruett N-vinyl-pyrrolidinone and UV-light. In particular, the UV-cross-linked HA hydrogel revealed excellent biophysical characteristics, a refractive index of 1.338 and good biocompatibility. Moreover, in an in vivo rabbit model, it was demonstrated that the cross-linked hydrogels remained inside the eye for at least 6 months. No inflammatory or toxic reactions and no lens opacifications were observed [To overcome the problem of rapid biodegradation of native HA, more recent approaches considered the use of cross-linked polymer gels , 10. Croobserved , 10.In this study, a vitreous substitute based on cross-linked thiolated HA was evaluated in order to determine whether in vivo biocompatibility testing could be performed. The thiol-modified polymer is able to build stable hydrogels by natural formation of disulfide bridges and thus does not require the addition of chemical cross-linkers or other manipulation. The generated hydrogel was characterized with respect to optical and rheological properties and subsequently tested as a vitreous substitute in two distinct ex vivo eye models in order to investigate short-term biocompatibility (in respect to ERG changes) and IOP development.For generation of an artificial VBS, a viscoelastic hydrogel was prepared from chemically derivatized thiol-substituted hyaluronic acid. The test item was a biodegradable, clear, viscous hydrogel, free of visible particles, homogenized and prepared as an injectable gel implant, which had been steam-sterilized. Formulations were prepared in physiological phosphate buffer  that contained 1% HA. Crosslinking was achieved by inducing disulfide bridge formation in the substituted thiol groups .A MCR101 rheometer equipped with a 50 mm cone-plate (CP50-1) measuring device  was used for rotational and oscillatory rheological analysis. The linear viscoelastic range was determined by amplitude sweep measurements for each sample separately and the determined deformation γ was adjusted accordingly for the following frequency sweep measurements (n = 5). Storage (G’) and loss modulus (G”) were determined and the ratio of viscous and elastic parts (Tan δ) were calculated accordingly using the RheoPlus software  .Before measurement, sample and device (Krüss refractometer AR 2008) were adjusted to 35°C ± 2°C. Double distilled water was measured as a control and the internal standard value adjusted accordingly. Approximately 1 g of hydrogel was applied to the prism and the refractive index (RI) was determined at 589 nm (n = 5). In compliance with ISO16672:2003, RI was also determined at 546 ± 10 nm using a Carl Zeiss refractometer (CZ11401) and an LED lamp emitting at 546 nm.The injection force of the manufactured hydrogel (5 ml glass syringe) was determined with a motorized device  by pressing the substance (12 mm/min) through a 20 G cannula (Geuder 33051). Measurements were performed in triplicate, and the respective mean value is reported as the result.A variation of the porcine anterior chamber model previously described by Johnson et al. , 13 was Half of a porcine eyecup was used to prepare tissue punches after 24 h of exposition . These samples were then used either for qRT-PCR or western blot analysis. Expression levels of Bax/Bcl–2, GFAP and PGP9.5 were analysed by qRT-PCR (n = 5). The amount of β-3 tubulin (n = 4) was assessed via western blot analysis. GAPDH was used as a control gene. Student’s t-test was used for statistical analysis (p<0.05). The standard deviation of the mean was calculated.2, 0.15 mM CaCl2, 1.5 mM NaH2PO4, 13.5 mM Na2HPO4 and 5 mM glucose with a final pH of 7.8, protected from light. The preparation was performed as described earlier [via two silver/silver-chloride electrodes placed on either side of the retina. After recording, the chamber containing a piece of retina was placed in an electrically and optically insulated box. A roller pump kept the superperfusion velocity at 1 ml/min, while the temperature was kept constant at 30°C. The superperfusion medium was pre-equilibrated and saturated with oxygen. After the retina was dark-adapted for 1 h at 30°C under constant superperfusion, the ERG was elicited at intervals of 5 min using a single white xenon flash for stimulation. A flash intensity of 6.3 mlx at the retinal surface was set using calibrated neutral density filters (Kodak Wratten Filter). Ten-microsecond durations of light stimulation were controlled with a timer . The ERG was filtered and amplified  using a Grass RPS312RM amplifier. With an analog-to-digital data acquisition board , the data were processed and transferred to a desktop computer (PC compatible).Bovine eyes were obtained directly postmortem  and transported in a serum-free standard medium containing 120 mM NaCl, 2 mM KCl, 0.1 mM MgClThe retina was superfused with serum-free standard medium and stimulated repeatedly until stable b-wave amplitudes were recorded. Stable responses were achieved after approximately 2 h of superperfusion. VBS was applied epiretinally, while superperfusion was stopped for 2 min. Subsequently, VBS was removed, superperfusion was restarted, and ERG recording continued for an additional 5 min. The superperfusion with standard solution was resumed for another 70 min to monitor b-wave recovery. The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave .To investigate the effect on the photoreceptor potential, the b-wave was suppressed by adding 1 mM aspartate to the nutrient solution before the actual testing started. Under these conditions, the influence of VBS on the photoreceptors was analysed. After recording a stable photoreceptor potential for 30 min, we proceeded as described above. A series of 5 independent experiments with different retinas was conducted. The recovery of a- and b- waves was compared to the corresponding amplitudes after application of VBS and at the end of the washout .For statistical analysis, JMP statistical software  was used. Data were calculated throughout as the mean and the standard deviation. Significance was estimated by Student´s t-test; p ≤0.05 was considered to be statistically significant.For generation of an artificial VBS, a viscoelastic hydrogel  with thiolated HA was prepared by induction of disulfide bridge formation. The generated VBS revealed a refractive index of 1.34 at 589 nm and 1.32 at 546 nm, respectively. Rheological analysis with hydrogel pre-sheared by ejection from a 5 ml syringe through a 20-gauge needle resulted in a storage modulus  of 128972 mPa and a loss modulus  of 5750 mPa, indicating the highly elastic nature of the generated VBS. This is also expressed by the Tan δ, which was accordingly calculated as 0.045 . As a thIn conclusion, the newly generated HA-based hydrogel revealed all basic physical characteristics required for its application as a vitreous substitute . ConsequFor evaluation of the effects of the generated artificial VBS on IOP, an explanted pig eye model was used. After a core vitrectomy including careful removal of the peripheral vitreous base and a fluid-air exchange, VBS was injected via a 20-gauge Weber-needle. The procedure was performed by two experienced vitreoretinal surgeons. The injection was performed manually without any problems. Continuous IOP measurements  revealedAfter injection of only BSS, an increase of 42% after 12 h was noted, and a 13% increase was observed after 24 h p>0.05, .The Bax/Bcl-2-expression level ratio as a marker for apoptosis did not show any significant difference between BSS-treated eyes set as 1.0 or eyes treated with VBS after 24 h of exposure . No signs of astrocyte or Müller cell activation were observed via qRT-PCR of GFAP expression in hydrogel-treated vs BSS-treated eyes, which were set as 1.0 . No significant differences in β-3–tubulin protein expression in hydrogel-treated versus BSS-treated were seen on western blotting , and no significant decrease of PGP9.5 mRNA expression  was observed, indicating that there had not been any loss of retinal ganglion cells.In addition to the effects of VBS implantation on IOP, compatibility of the hydrogel with retinal function was investigated. For this purpose, an ex vivo bovine retina model was used, and the influence of applied VBS on electrophysiological parameters was studied. ERGs (a- and b-waves) taken from the superfused retina before application were compared to measurements shortly after VBS application and at the end of a 70-min washout period. Five minutes after exposing the retina for 120 seconds to VBS, a 9% reduction of the b-wave amplitude was observed, which was, however, not statistically significant p>0.05, . SimilarDuring the testing of the photoreceptor potential measured by the a-wave amplitude, an increase of 30.4% directly after VBS application was noted, which was again not statistically significant. At the end of the observation period, the value was increased by 23.9%, which also did not reach statistical significance p>0.05, . Mean vaFacing the demand for distinct vitreous substitutes with better biocompatibility, less vision disturbance and defined stability , a newlyThe moderate, statistically non-significant increase in IOP determined from the porcine anterior chamber model corresponds well with data from in vitro swelling assays, indicating a maximum weight increase (wet versus dry) of the tested hydrogel by 50% after 48 h. This swelling is comparable to existing hydrogels and lower than existing tamponades such as silicone oils ,18–20.The used ex vivo pig eye model is a variation of the well described anterior chamber model established by Johnson et al.  and modiThe isolated bovine retina, which shows significant analogies to the human retina, is a sensitive and well-evaluated tool for short term pharmacological testing and, in this respect, has shown comparable results to human isolated organ cultures ,24. A poThe obtained results demonstrated that neither a-wave nor b-wave amplitudes were affected significantly by short-term application of the hydrogel. The high standard deviation noticed shortly after exposition of the retina with the vitreous substitute is caused by the experimental setup and has been discussed earlier , 27. ThuAlthough ERG recordings from superfused retinae are very valuable for a functional analysis on cellular level, longer exposition periods than 12 h cannot be tested under these highly standardized conditions. Moreover, the exposure time of the hydrogel in the present experiments was limited to 120 seconds due to its highly viscous nature, which did not allow for continuous superperfusion of the system. Since ex vivo retinal viability is strongly dependent on a sufficient supply of oxygen and nutrients, longer exposure times were not feasible. Moreover, the model of the superfused retina primarily provides information about the integrity of the outer retina; the effect of VBS on the inner retina, e.g., ganglion cells, cannot be sufficiently evaluated by this means. Thus, biocompatibility testing of the new VBS prototype beyond the 2-min exposure period will depend on an in vivo model. To overcome this shortcoming in our setup, qRTPCR and western blot analysis were performed over an exposure period of 24 h using PV, indicating good biocompatibility. It is arguable that the clinical exposure time is considerably longer; therefore, in vivo biocompatibility experiments are recommended. The presented ex vivo data, however, underline the potential of this HA-based VBS and PV for vitreoretinal surgery.In summary, the manufactured thiolated HA-based hydrogel showed highly favourable biophysical characteristics in the explored ex vivo models, strongly suggesting its further application as a vitreous substitute in vivo.


Background: Immune checkpoint inhibitors (ICIs) are now an important option for more than 14 different cancers. Recent series case reports have described that ICIs are associated with new-onset diabetes in patients, yet the definitive risk is not available. We thus performed a meta-analysis of randomized controlled trials (RCTs) to assess the incidence and risk of developing new-onset diabetes following the use of ICIs.Methods: The PubMed, EMBASE, Cochrane Library databases, and ClinicalTrials.gov for RCTs were searched. Statistical analyses were performed using STATA 15 and R language. Fifty-two RCTs were included, and 12 did not report any events of ICI-associated diabetes.Results: A meta-analysis of 40 trials was performed, which reported at least one diabetes-related event among 24,596 patients. Although specific diabetes-related events were rare, compared with the placebo or other therapeutic strategies, the rates of serious hyperglycemia , diabetes , all-grade T1D , and serious-grade T1D  were increased with ICI drugs. Subgroup analysis according to the type of control, type of ICIs, and the combination mode suggested that ICIs plus conventional treatments significantly decreased the risks of diabetes and serious-grade hyperglycemia. There was little heterogeneity across the studies in all results except hyperglycemic events, which in part was attributable to data from everolimus-based control group.Conclusions: New-onset diabetes is uncommon with ICIs but the risk is increased compared with placebo or another therapeutic strategy. However, more studies are warranted to substantiate these findings across ICIs. Immune checkpoint inhibitor (ICI)-based treatments that block molecules such as programmed cell death protein 1 (PD-1), PD1 ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) have emerged as powerful weapons in a growing number of cancers . CurrentAmong these irAEs, new-onset diabetes is receiving increased attention, as more evidence suggests the recognition of diabetes-related adverse events in patients with cancers who are treated with ICIs. A marked increase in reporting diabetes has also been seen since 2017 by analyzing the World Health Organization’s database of individual case safety reports . These oGiven the dramatic growth in the number of clinical trials testing ICI agents and their clinical benefits in the increasing list of cancer types and negative influence on life quality caused by diabetes if not promptly recognized, we performed a meta-analysis of randomized controlled trials (RCTs) with ICIs in patients with cancer and evaluated the incidence and risks of diabetes-related adverse events compared with placebo or another therapeutic strategy.Scientific literature searches were performed in three databases  from the inception of all searched databases to March 2019. Relevant text words and medical subject headings that consisted of terms including ‘phase’ and the individual drug names . Two of three authors reviewed and discussed the potential full text. Any disagreements were resolved by consensus with all three .Data from each study that met the inclusion criteria were independently extracted by two of the three authors . Any disagreement was resolved by consensus with all three. The retrieved data included author name, year of publication, trial characteristics , patient characteristics , the sizes of the intervention and control groups, ICI treatment, dose, and the outcomes of interest. We detected new-onset diabetes following treatment with ICIs using the following terms: hyperglycemia, diabetes mellitus (DM), type 2 diabetes (T2D), and type 1 diabetes (T1D). For data extracted from ClinicalTrials.gov, adverse events were reported as either serious or other; for data from published reports, we identified grades 3–5 as serious and grades 1–2 as other, according to Common Terminology of Clinical Adverse Events categorization. If data were available for both sources, we prioritized data from sources where the data were more complete. If a published study did not report diabetes-related adverse events, and the corresponding registry trial from ClinicalTrials.gov reported did, we included the registry report. For multiple reports of the same trial, only the most completely reported data were used. The quality of the included studies was independently assessed using the Cochrane Risk of Bias Tool. We considered all trials at unclear risk of incomplete outcome data and selective reporting bias as these studies were not designed primarily to assess adverse events.I² statistic and P value were used to examine heterogeneity across trials for each outcome. An I² statistic of 0–25%, 26–75%, and 76–100% was regarded as indicating low, moderate, and high heterogeneity, respectively. A P value of less than or equal to 0.05 was defined as significant heterogeneity. If a study included more than one intervention group (e.g. different doses or different types of ICI), we separately compared each intervention group with the control group, where the number of patients or events in the control group would be doubled. Sensitivity analyses were performed excluding an everolimus-controlled study, which was known to cause diabetes-related adverse events, to understand the reasons for the high likelihood of differences. We conducted subgroup analyses to examine studies according to the type of control group (chemotherapy vs. immunosuppressive drug vs. targeted therapy vs. placebo), the mode of intervention treatment (monotherapy vs. add-on therapy), and the type of ICI (PD-1 vs. PD-L1 vs. CTLA4 vs. combination of ICIs). Evidence of publication bias was assessed using Egger’s and Begg’s test in addition to funnel plots, and significant publication bias defined as a P < 0.1. All statistical analyses were conducted with STATA, version 15.The estimated event rates in the intervention group are calculated as the total number of patients with a given adverse event divided by the total number at risk. Data were transformed using the Freeman-Tukey Double Arcsine transformation to calculate event rates. This statistical analyses were performed using R statistical software . For risk outcome, we pooled trials and calculated odds ratios (ORs) and their associated 95% confidence intervals (CIs) in the intervention group compared with the control group based on the number of patients with a given adverse event and sample size. Given the low rates of adverse events, we used Peto’s method to pool effect estimates across studies. The Our search from the PubMed, EMBASE, and Cochrane Central Register databases yielded a total of 8,596 potentially relevant reports  and 0.28% , respectively. The rates of hyperglycemia events differed by the type of ICI and tumor. In particular, patients treated with ICI combination therapy were more likely to report hyperglycemia: 3.37% for all-grade hyperglycemia events, 0.47% for serious-grade hyperglycemia. Patients with RCC showed a trend toward higher rates of both all-grade and serious-grade hyperglycemia, with rates of 6.82% and 0.66%, respectively. High dose of ICIs was not associated with high rates of hyperglycemia events , five cases of T2D occurred in 3,117 patients (raw event rate 0.16%), and 17 cases of all-grade T1D occurred in 3,899 patients (raw event rate 0.44%), and 15 cases of serious-grade T1D events occurred in 3,603 patients (raw event rate 0.42%).To assess the relative rate of ICI-associated diabetes compared with those in control arms, we calculated the OR of developing diabetes in the RCTs. Pooling the data of these studies showed that patients treated with ICIs were at higher risk for serious-grade hyperglycemia . Subgroup analysis based on the mode of treatment (monotherapy vs. add-on therapy) suggests that add-on therapy decreased the risk of ICI-associated diabetes, with OR 1.77 for DM, 1.31 for serious-grade hyperglycemia, 0.58 for T2D, and 5.83 for T1D (P = 0.994), all-grade hyperglycemia (Egger’s test P = 0.128), serious-grade hyperglycemia (Egger’s test P = 0.325), T2D (Egger’s test P = 0.310), all-grade T1D (Egger’s test P = 0.300), and serious-grade T1D (Egger’s test P = 0.334) (I² = 0.0%). However, we noted substantial heterogeneity for the outcome of all-grade hyperglycemia (I² = 88.2%), which was considerably reduced in the analyses of data excluding the everolimus-controlled study (I² = 8.0%).The funnel plot and statistical test showed no evidence of publication bias for DM .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cholesteric liquid crystal (CLC) has attracted intensive attention due to its ability to form a periodic helical structure with broad tunability. CLC gratings in open systems are especially promising in sensing and micromanipulation. However, there is still much to learn about the inherent mechanism of such gratings. We investigate the light-driven rotation and pitch-tuning behaviors of CLC gratings in semi-free films which are formed by spin-coating the CLC mixtures onto planarly photoaligned substrates. The doped azobenzene chiral molecular switch supplies great flexibility to realize the continuous grating rotation. The maximum continuous rotational angle reaches 987.8°. Moreover, dependencies of light-driven rotation and pitch tuning on the dopant concentration and exposure are studied. The model of director configuration in the semi-free film is constructed. Precise beam steering and synchronous micromanipulation are also demonstrated. Our work may provide new opportunities for the CLC grating in applications of beam steering, micromanipulation, and sensing. P is defined as the length of one turn of the helical structure. When a proper electric field is applied, the helical axis turns over and subsequently a CLC grating is resulted  the ability to form periodic structure with broadly tunable feature size. The generation of the CLC grating is strongly dependent on the anchoring condition. There are three different modes. In the homogeneous cell ,3,4,5,6,resulted . CLC graresulted . For theresulted . All theBy spin-coating a CLC mixture on a unidirectionally-aligned substrate, a semi-free film is obtained. In this case, the substrate provides a planar anchoring and the air induces a near-vertical anchoring . TherefoHere, CLC gratings are formed by spin-coating CLC mixtures, doped with a left-handed azobenzene chiral molecular switch onto planarly photoaligned substrates. Due to the photoinduced isomerization of the chiral molecular switch, the helical twisted power (HTP) of CLC can be continuously modified, resulting in the change of layer configuration inside the semi-free film. We investigate the dependencies of light-driven rotation and pitch tuning behaviors of CLC gratings on the dopant concentration and the exposure, respectively. The director configuration in the semi-free film is simulated. On the basis of the new understanding of themechanism and rules of the CLC grating, applications including beam steering and micromanipulation with distinctive characteristics are demonstrated. This work may supply new insights into the promising semi-free material system and inspires more fantastic applications.−4 J/m−2, which is comparable to that of rubbed polyimide film [Photoalignment avoids any mechanical damage, electrostatic charge, and dust contamination; thus, it is suitable for high-quality LC alignment . Here, aide film ,32,33.trans-form to bent cis-form structures upon blue light irradiation. The cis-form can be stable for several days, while heating or green light exposure will accelerate the cis-to-trans isomerization [HTP,C % and ee are the concentration and the conformational change of the chiral molecular switch, respectively [A left-handed azobenzene chiral molecular switch of ChAD-3c-S (BEAM) is adopted for phototuning of the CLC helical structure. It features two azo linkages, which can isomerize from the rod-like rization ,35,36. Tectively . The CHA2) were ultrasonically bathed, UV-ozone cleaned, and then spin coated with SD1. After curing at 100 °C for 10 min, the substrates were photoaligned by a linearly polarized UV light to achieve unidirectional alignment. Then, the ChAd-3c-S-doped CLC mixture was spread onto the substrate at 50 °C and spin-coated at 1000 rpm for 30 s; thus, the semi-free film was obtained. All samples were fabricated under the same condition.Glass substrates (1.5 × 2 cm2). The diffraction patterns were captured by digital camera .The semi-free films were observed under a transmission polarized optical microscope (POM)  with a weak light intensity. The photostimulating process was performed under the reflection mode of POM through a bandpass filter  is ~2.3 μm , which is measured by the traditional Grandjean–Cano method [Λi) was measured as ~5.2 μm. Under the irradiation of blue light, the chiral molecular switch isomerizes, modifying the HTP and subsequently changing the layer configuration. As presented in ϕ) are 135° and 1122.8° , respectively. That means that the total continuous rotational angle αt of 987.8° was achieved, which is larger than any reported results [After the CLC mixture with 1 wt % ChAD-3c-S was spin coated, the CLC grating  was obtained in a thin semi-free film a. Such Co method . The gra results . In the Λ and ϕ on the dopant concentration (labeled by 1/Pi). The initial orientation ϕi was measured once the CLC grating is formed, which is in the range of 0° to 180° as depicted in the inset of ϕend was determined when the grating was disappearing π , which is clearly exhibited in αt is directly proportional to 1/Pi . Sixth, the value of Λi/Pi is in the range of 1.8 to 2.3  should be the same. In this case, D is equal to aPend for all the semi-free films, where a is a constant describing the residual amount of the helical pitch. This assumption could be verified by the same ends of the cholesteric helixes  are constructed (D = 4.5Pi_0.43 (αt_0.43/360°. There are still 1.75 residual pitches left at the end state.We simulate the director configuration of the semi-free film based on Baudry’s model . Accordistructed . The sem5Pi_0.43 , and oth5Pi_0.43 . Figure Pi = 0.43 μm−1 to characterize the beam steering performance of the CLC grating. Circular polarization is adopted to avoid the influence of polarization-dependent effect on the diffraction efficiency. A 633 nm probe beam is normally incident to the sample of 1/ZSM-5 zeolite microparticles are chosen for the demonstration of micromanipulation. The typical length of the long axis is ~12.6 µm and the aspect ratio is ~1.32. The microparticles of ZSM-5 zeolite are spattered onto the surface of the semi-free film, and then their rotation behaviors are monitored. HTP, the CLC grating rotates along with the unwinding cholesteric helixes. This supplies great flexibility to the realization of continuous grating rotation. Dependencies of the light-driven rotation and pitch tuning on the dopant concentration and exposure are studied. The maximum rotational angle reaches 987.8°. The end grating orientations of all samples are fixed at the same direction. On the basis of the new understanding on the mechanism and rules of CLC gratings, the layer configuration of the semi-free film is simulated. The model depicts the director configuration of the sample and vividly reveals the relationship between the helixes unwinding and grating rotation. Distinct superiorities of the CLC gratings and applications including beam steering and micromanipulation are also demonstrated. Phenomena such as diffraction orders rotation on fixed concentric circumferences and synchronous rotation of microparticles with the CLC grating are reported for the first time. This work may offer new insights into the promising semi-free structured materials and inspire more fantastic applications.In summary, we investigate the light-driven rotation and pitch tuning of CLC gratings in semi-free films. Due to the continuous photoinduced modification of


Atrial fibrillation (AF) is one of the most common types of arrhythmias and often leads to clinical complications. The objectives of this study were to offer insights into the metabolites of AF and to determine biomarkers for AF diagnosis or prediction. Sixty atrial appendage samples  and 163 plasma samples  from 49 AF patients and 116 non-AF patients were subjected to liquid chromatography positive ion electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) metabolomics analysis. Consequently, 24 metabolites in atrial appendage samples and 24 metabolites in plasma samples were found to reflect metabolic differences between AF and non-AF patients (variable importance in projection (VIP) ≥ 1, P ≤ 0.05). Five identical metabolites including creatinine, D-glutamic acid, choline, hypoxanthine, and niacinamide  in atrial appendage and plasma samples were considered prominent features of AF patients, and the D-glutamine and D-glutamate metabolic pathway was also identified as a feature of AF patients. Finally, in plasma samples, the combination of D-glutamic acid, creatinine, and choline had an AUC value of 0.927  and displayed 90.5% sensitivity and 83.3% specificity; this group of metabolites was thus defined as a combinational biomarker for the recognition of AF and non-AF patients. In recent decades, studies have indicated that control of traditional risk factors for cardiovascular disease may not reduce AF to an appreciable extent because there are some other well-established specific risk factors for AF3. These factors include age; arterial hypertension; congestive heart failure, including heart failure with impaired or preserved left ventricular systolic function; myocardial infarction4; valvular heart disease (VHD); and diabetes mellitus5. There are also emerging risk factors for AF, such as subclinical hyperthyroidism, obesity, chronic kidney disease, obstructive sleep apnea, heavy alcohol use, and even high-level endurance training, but the evidence does not clearly indicate that eliminating one or more of these risk factors protects against AF recurrence7.Atrial fibrillation (AF) is the most common type of arrhythmia and has a substantial effect on individual morbidity and mortality as well as on healthcare expenditure. AF has a prevalence of 0.7% in individuals between the ages of 55 and 59 years; the incidence rises to 17.8% in individuals aged 85 years and over8. Untargeted  and targeted mass spectrometry-based metabolomics are the main methodologies for metabolite recovery and identification9. These methodologies are based on gas chromatography or liquid chromatography mass spectrometry (GC-MS or LC-MS), which reveals concomitant changes in metabolic pathways including glycolysis, fatty acid β-oxidation, and lipid biosynthesis8.Metabolomics is routinely applied as a tool for biomarker discovery to profile metabolites of biofluids, cells, and tissues12 or the initiation and maintenance13 of AF have previously been researched, including inflammatory factors14, prothrombotic markers15, and microRNAs17. A nuclear magnetic resonance (NMR) metabolomics technique was used to analyze atrial profibrillatory remodeling in a ventricular-tachypacing (VTP)-induced congestive heart failure (CHF) model in dogs1 and in atrial appendage tissues in AF patients18, but the results have not been replicated. The NMR technique has its own disadvantages, including low analytical resolution and insensitivity in comparison to mass spectrometry, although it is very powerful in metabolite qualification20. LC-MS has been deemed to be a fast, high-resolution separation technique similar to ultra-high-performance liquid chromatography (UHPLC), and LC-MS enables the detection of thermolabile molecules19. Since 2008, LC-MS has been popular in cardiovascular disease metabolomics analysis21. In this study, untargeted LC-MS was utilized to investigate the different metabolites in atrial appendage and plasma samples. To distinguish altered metabolites in the samples and to discover metabolic biomarkers for AF diagnosis or prediction, comparative investigations were performed between AF patients and non-AF patients.Biomarkers associated with the recurrence and prognosisIn this study, a total of 165 cardiovascular disease patients were enrolled, including 49 AF patients (AF group) and 116 non-AF patients (non-AF group). There were fewer male patients in the AF group than in the non-AF group (27 (55.10%) vs. 89 (76.72%), P < 0.001). The heart rate (HR) of AF group was higher than that of non-AF group , and the left atrial diameter (LAD) was also larger in the AF group than in the non-AF group , but the systolic pressure was lower in the AF group than in the non-AF group . Furthermore, the creatinine clearance rate (Ccr) of the AF group was lower than that of the non-AF group . In addition, the heart failure history of AF group was higher than that of non-AF group (19 (38.78) vs. 23 (19.83), P = 0.011), and the major adverse cardiac and cerebrovascular event (MACCE) incidence were higher in the AF group than in the non-AF group (11 (22.45) vs. 9 (7.76), P = 0.008, respectively) either. Moreover, the patients were split into distinctly different groups based on the New York Heart Association (NYHA) classes of heart failure , II , III , and IV  for the AF group vs. I , II , III , and IV  for the non-AF group, P < 0.001). The other clinical characteristics (Table 2 ellipse). Validation of the models for the atrial appendage samples was performed. The S-plot of OPLS-DA is shown in Fig. More than 80% of the metabolites in the LC-MS raw data with null data were deleted. The rest of the null data were replaced with 1/2 of the minimum signal. The acquired LC-MS raw data were normalized. A PCA model helped us delete a few outlier samples; the PCA scores were plotted to determine the aggregation and dispersion of samples Fig. . A PLS-DAmong the 24 metabolites for the atrial appendage samples, 8 metabolites  had VIP values above 3.0, which indicated a strong difference between the AF group and the non-AF group. Eleven metabolites  were significantly increased in the AF group relative to the levels in the non-AF group, whereas the 13 remaining metabolites  were all decreased  had VIP values above 3.0, which indicated a strong difference between the AF and non-AF groups. Seven metabolites  were significantly decreased in the AF group relative to the levels in the non-AF group, whereas the 17 remaining metabolites -2-methylmalate/(R)-2-methylmalate/2-hydroxyglutarate, acetylcholine/deoxycarnitine, N6,N6,N6-trimethyl-L-lysine, ergothioneine, and N-acetyl-DL-serine) were all increased  had the same tendency to increase in all samples, 1 metabolite in AF patients (creatinine) increased in atrial appendage samples but decreased in plasma samples, and 3 metabolites in AF patients  declined in atrial appendage samples but rose in plasma samples. To determine which metabolites caused segregation, a VIP value ≥ 1.5, a P value < 0.01, and a false discovery rate (FDR) < 0.05 were usedHeatmaps were created through HCA to provide intuitive visualizations of the differences in the patient metabolites between the AF and non-AF groups; comparisons of the atrial appendage samples and the plasma samples of the non-AF group and the AF group are shown in Fig. KEGG analysis of the differential metabolites in the atrial appendage samples and plasma samples are shown in Tables 23, so we filtered out 5 metabolic pathways  in the atrial appendage samples and 3 metabolic pathways  in the plasma samples. After comparing the two groups, we determined the ‘D-glutamine and D-glutamate metabolism’ pathway to be the main canonical pathway in AF, which is consistent with the fact that D-glutamic acid is a prominent feature of AF in cardiovascular disease patients24.An impact value greater than 0.10 indicates that the altered pathways clearly affect AFDifferential metabolite correlation heatmaps for the atrial appendage samples and the plasma samples are shown in Fig. Among the plasma metabolites, the 16 increased metabolites (from hypoxanthine to D-glutamic acid on the right side) were nearly positively correlated with each other but were negatively correlated to the last 7 metabolites , which were decreased and were positively correlated with each other. The metabolites 3-methyl-2-oxovaleric acid/2-ketohexanoic acid and glutarate semialdehyde/alpha-ketoisovaleric acid as well as D-glutamic acid and N-acetyl-DL-serine showed positive degrees of correlation reaching 0.8. However, glycerophosphocholine was negatively correlated (degree of correlation: −1.0) with creatinine Fig. .After the differential metabolite screening, Z-scores were used for statistical analysis  and one cardinal pathway (‘D-glutamine and D-glutamate metabolism’) had been filtered, indicating that the metabolite D-glutamic acid is critical in the pathophysiological mechanisms of AF. Thus, we drew ROC curves based on D-glutamic acid. The ROC curves are shown in Fig. Among the metabolites of the atrial appendage samples, metabolite C  showed a high diagnostic effectiveness Fig. . MetabolLikewise, among the metabolites of the plasma samples, metabolite A had AUC values less than 0.7, metabolites B/C/D/E and the combinational markers AC/AD/AE had higher AUC values between 0.7 and 0.8 (P < 0.001), and metabolites AB/ABD/ABE/ACD/ACE/ADE had AUC values greater than 0.8 but less than 0.9 (P < 0.001) Fig. . However3. Biomarkers derived from the blood may reflect the current disease state or the prognosis of diseases. Accompanied by the development and progression of AF, metabolite biomarkers might reflect the pathophysiological mechanisms of AF. Mayr et al.18 performed an untargeted NMR-based metabolomic analysis to demonstrate that both β-hydroxybutyrate and its ketogenic amino acid as well as glycine were increased in AF patients’ atrial myocardial samples. This NMR-based metabolomic analysis focused on postoperative AF. However, the NMR technique has its own disadvantages, as previously mentioned. Furthermore, an ideal innovative biomarker for diagnosis or prognosis should be able to be measured through minimally invasive techniques, should have high sensitivity and specificity, and should have good operability and repeatability to be feasible for the patients. Therefore, biomarkers in the blood are worth continued consideration.After decades of research and therapeutic intervention for AF, scientists are still striving to understand the causes and mechanisms of the condition. Biomarkers derived from blood, such as markers of coagulation, renal function, inflammation, myocardial injury, and cardiovascular stress, have been associated with clinical eventsIn this study, 60 atrial appendage samples and 163 plasma samples from 165 cardiovascular disease patients (49 AF patients and 116 non-AF patients) were subjected to LC-MS/MS-based metabolomics to investigate whether AF alters the metabolism of cardiovascular disease patients. Moreover, untargeted metabolites in tissue and plasma were compared to determine whether the differential metabolites could be potential biomarkers for AF diagnosis or prediction. Twenty-four metabolites from plasma samples and 24 metabolites from atrial appendage samples were screened for their ability to distinguish AF from non-AF patients  deficiency, which leads to gout, hyperuricemia, and Lesch-Nyhan syndrome30. Researchers have demonstrated that hypoxanthine can cause endothelial dysfunction through oxidative stress-induced apoptosis29. To a certain extent, our results hint that hypoxanthine imbalance can result in AF. Additionally, glycerophosphocholine has been reported to accumulate in human aortic tissue and plasma in response to early atherosclerosis18; in our study, glycerophosphocholine was increased in the atrial appendage samples and plasma samples of AF patients. D-glutamic acid belongs to the group of D-amino acids, which are metabolized only by D-aspartate oxidase (DDO) in mammals31 and are now thought to be naturally occurring physiologically active substances and biomarkers33. D-glutamic acid might activate transporter-associated Cl− conductance and has been reported to regulate neuronal transmission34. Kazuaki et.al. demonstrated that in 2-DM patients, preserved left ventricular ejection fraction reduced heart rate recovery and was associated with AF35, illustrating that autonomic neuropathy seems to be involved in the pathogenesis of AF. Thus, D-glutamic acid may influence the autonomic nervous system, resulting in AF. Furthermore, our results from the LC-MS/MS metabolomics analysis regarding hypoxanthine, D-glutamic acid, and niacinamide suggest that these metabolites might be novel biomarkers associated with AF, but this conclusion should be closely inspected.Eight identical metabolites were identified between the atrial appendage samples and the plasma samples before the comparison of expression in this study  in the samples from the two groups were filtered  was selected from the samples of the two groups. The fact that the ‘D-glutamine and D-glutamate metabolism’ pathway had an impact value greater than 0.10 (0.33) suggests that the ‘D-glutamine and D-glutamate metabolism’ pathway and the metabolite D-glutamic acid are prominent features of AF in cardiovascular disease patients. The KEGG analysis offered us a foundation on which to determine that the metabolite D-glutamic was indispensable to AF.KEGG analysis Tables  and 3 an36. An area under the curve (AUC) greater than 0.7 represents very high diagnostic effectiveness, and the effectiveness is particularly good when the AUC value is greater than 0.937. ROC curves based on D-glutamic acid were drawn to explicitly determine which metabolites can definitely diagnose AF in patients. Our results revealed that the combinational marker comprised of D-glutamic acid, creatinine, and choline had an AUC value of 0.927 , while the diagnostic effectiveness of this combinational biomarker achieved 90.5% sensitivity and 83.3% specificity, demonstrating that its ability to predict AF in plasma samples was satisfactory  curves have been successfully applied to evaluate the diagnostic accuracy of biomarkers in many studiesory Fig. . In thisRelated investigations were conducted under the approval of the Ethics Committees of the Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College . All participants provided written informed consent at the time of enrollment. Two groups of cardiovascular disease patients, an AF group (49 AF patients) and a non-AF group (116 non-AF patients), were included in the present study. Sixty atrial appendage samples and 163 plasma samples were collected from the patients at Fuwai Hospital from May 2015 to March 2016. Atrial appendage samples were obtained from cardiovascular disease patients who had surgical treatment. All methods were performed in accordance with the relevant guidelines and regulations. The datasets and methods, including the methods of data acquisition and analysis, are available from the corresponding author upon reasonable request. The workflow of our study is shown in Fig. g for 10 min at 4 °C, and the clear supernatant was transferred to a new Eppendorf tube. All plasma samples were promptly frozen in a −80 °C freezer until analysis. Sample preparation followed a method that has been described in recent literature38. Frozen samples were thawed at room temperature, extracted in 75% aqueous methanol containing 0.1% formic acid, and analyzed by untargeted LC-MS as follows.Atrial appendage samples were collected during surgery following surgical principles. The atrial appendage was cut into small pieces, transferred to a clean tube, and immediately stored at −80 °C until use. The blood samples were collected in ethylenediaminetetraacetic acid (EDTA)-coated anticoagulation tubes the morning after the patients were hospitalized. The collected blood was immediately centrifuged to isolate the plasma at 3,000 × Methyl alcohol, acetonitrile, and all other materials and chemicals were of HPLC grade and were obtained from Sigma-Aldrich Co. (USA). Ultrapure water (Milli-Q) was obtained from a Milli-Q Gradient Direct 16 system . Sample analysis was performed using an electrospray ionization (ESI) TSQ Vantage triple quadrupole quantum mass spectrometer  with a Surveyor Autosampler combined with an Agilent 1200 Series HPLC System  equipped with Xcalibur 3.0 software .39. PLS-DA and OPLS-DA were performed to clarify the groups among clusters40, which was particularly helpful for detecting and removing abnormal samples to improve the accuracy of the model. Finally, Student’s t-test (P < 0.05) was combined with the variable importance in projection (VIP) value41 of the first principal component or the data were visualized using the S-plot42 in OPLS-DA to search for distinct metabolites.Raw data acquired from the HPLC-MS systems were converted into databases with equal quality and retention time peak alignment. Then, peak discrimination, total peak area normalization, filtering, alignment, and matching were performed. Afterward, annotated metabolites were put through SIMCA-P 13.0  for statistical analysis. Principal component analysis (PCA) was conducted using the Pareto model as an unsupervised analysis to view the clustering trend while removing outlier samples. Later, partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were employed as a supervised analysis using a permutation test (200 permutations) to prevent overfitting of the PLS-DA modelhttps://cran.r-project.org/web/packages/pheatmap/index.html). Cluster analysis or clustering is always used to determine the metabolic patterns of metabolites under different experimental conditions. Hierarchical clustering analysis (HCA) of samples in the heatmap was performed using the average linkage clustering method and Euclidian distances. Metabolites with the same metabolic pattern might possess the same function or participate in the same metabolic process or cellular pathway.Heatmap analysis was used to further study the relationships between metabolites and samples, where the intent was to discern similar metabolic profiles among samples or identical metabolic activity among metabolites. Heatmaps were created using the pheatmap R package  in R statistical software. MMCA offers a new insight into metabolomics. For instance, similar trends in variation among metabolites indicate positive correlations, while different trends in variation indicate negative correlations. When the linear correlation of two metabolites is enhanced, the correlation coefficient tends toward 1 or −1 for a positive correlation or a negative correlation, respectively. The correlation matrix was calculated through the cor function in R (v3.1.3) software (https://www.r-project.org/). The metabolite correlation heatmap was drawn based on the correlation coefficients.Additionally, metabolite-metabolite correlation analysis (MMCA)Standard scores, also called Z-scores, which are used to identify distinct metabolites, can evaluate the relative contents of the metabolites in each sample. The formula for the Z-score is z = (x − μ)/σ .44 (http://www.genome.jp/kegg/) is a database resource that integrates genomic, chemical, and systemic functional information. KEGG analysis was used to analyze the interrelationships of the metabolites in the metabolic pathways. In addition, enrichment analysis was performed with MetaboAnalyst 3.0 , a comprehensive server for metabolomic data analysis, including three functional analyses: enrichment analysis, pathway analysis, and integrated pathway analysis45. MetPA is a part of MetaboAnalyst that is mainly based on the KEGG metabolic pathways46. MetPA uses pathway enrichment analysis and pathway topological analysis to identify the potential metabolic pathways interfered with by biological perturbation. The topological analysis selected in this study was ‘relative betweenness centrality’. Furthermore, MetPA can analyze the related metabolic pathways of different metabolites between two groups through hypergeometric tests.The Kyoto Encyclopedia of Genes and Genomes (KEGG)Statistical analysis was performed with SPSS 22.0 software. Continuous variables were expressed as the means ± S.D., and categorical variables were presented as counts and percentages (%). Continuous variables were calculated by nonparametric tests or t-tests, categorical variables were analyzed using chi-square or Fisher’s tests, and multilevel data analysis was performed with the Mann-Whitney U test. A two-tailed P value < 0.05 was considered statistically significant. ROC curves and AUCs were calculated in SPSS 22.0 software , but the combinational marker ROC curves first required binary logistic regression.Supplementary Information


The SPFS performance is characterized in terms of acoustic frequency, amplitude responses, and sensitivities that are compared with those of a 1-mm diameter piezoelectric hydrophone used as a reference.We demonstrate a side-polished fiber-optic ultrasound sensor (SPFS) with a broad frequency bandwidth (dc–46 MHz at 6-dB reduction) and a wide amplitude detection range from several kPa to 4.8 MPa. It also exhibits a high acoustic sensitivity of 426 mV/MPa with a signal-to-noise ratio of 35 dB and a noise-equivalent pressure of 6.6 kPa (over 1–50 MHz bandwidth) measured at 7-MHz frequency. The SPFS does not require multi-layer-coated structures that are used in other high-sensitivity optical detectors. Without any coating, this uses a microscale-roughened structure for evanescent-field interaction with an external medium acoustically modulated. Such unique structure allows significantly high sensitivity despite having a small detection area of only 0.016 mm Optical ultrasound sensors have been actively studied to achieve high sensitivity and broad acoustic frequency bandwidth. It has been demonstrated that a variety of interferometric sensors have such characteristics. For example, polymer microring resonators ,2,3,4, FFor optical measurements of high-amplitude acoustic pressure, fiber-optic sensors with a flat cleaved edge have been widely utilized without any coatings have been used instead ,17,18,19A side-polished fiber (SPF) has provided a useful platform for a variety of optical sensors based on surface plasmon resonance (SPR) ,24,25, BWe demonstrate a side-polished fiber sensor (SPFS) for ultrasound detection with a frequency bandwidth up to 46 MHz, an amplitude dynamic range of several kPa–4.8 MPa, an acoustic sensitivity of 426 mV/MPa at 7 MHz, and a noise-equivalent pressure (NEP) of 6.6 kPa (over 1–50 MHz bandwidth) obtained at 7 MHz. Our SPFS has a particular optical architecture with the upper cladding region above the core entirely removed. Its partially polished core with microscale-roughened surface is exposed directly to an external medium. There is no additional coating on the polished region, making the structure mechanically robust and reliable against high-pressure amplitudes of several MPa. The SPF works as a narrow line form (8 μm) of an acoustic sensor based on evanescent-field interaction along the polished fiber region, enabling significantly higher sensitivity than other non-coated optical sensors. in/Pout) were 31 and 22.6 dB, respectively.A single-mode optical fiber  was mounted on a longitudinal groove (V-shaped) formed in a quartz block and then fixed using a UV-curable epoxy. Here, the groove was slightly bent with 250-mm radius of curvature, resulting in a small bent angle of α  probe laser beam  as an input, as shown in Here, the CNT-PDMS transmitter was designed and fabricated to have a frequency bandwidth of <30 MHz, which is relatively lower than the one reported elsewhere . Howeverin. Here, the angle of incidence (85°) is greater than the critical angle at the core/water interface (θC = 67°). After passing through region II, it couples back to the non-polished region (region III) connected to the photoreceiver, which is shown as Iout.The optical fiber can be divided into three parts along the longitudinal direction as demonstrated in s) and mode mismatch between regions. Such evanescent field interaction and thus the sensing process occurs over the entire volume of roughened structure, which is indicated by the thick arrow at the top of In the SPFS, the evanescent field of core mode interacts with the refractive index modulation of water in region II, although there are high optical losses via scattering (IWe compared acoustic frequency characteristics of SPFS (=SPF1) and PVDF hydrophone under the same measurement conditions. For broadband frequency characterization, we first used the Cr transmitter capable of duplicating an incident Gaussian shape of laser pulse into an acoustic pulse. This generates narrow ultrasonic pulses (~7 ns) with a bandwidth from dc to 70 MHz (at 6-dB reduction) which are suitable to find a detector output close to an impulse response . Figure Next, we used the CNT-PDMS transmitter as a source to compare the detector responses. It was significant to use CNT-PDMS transmitters because they are high-frequency and high-amplitude sources widely utilized for a variety of all-optical ultrasound transducers for imaging and therapy ,30,31. Fin) as demonstrated in in = 10–120 mW. The SPF output strength was limited by the maximum-available power of the probe laser source (120 mW).From the CNT-PDMS, the measured peak pressure values were consistently higher for the SPFS as the input pressure increased as demonstrated in For sensitivity evaluation, we used two calibrated piezoelectric transducers with center frequencies of 3 and 7 MHz as acoustic sources, as demonstrated in 2 calculated from 8.3-μm core thickness and 2-mm length. This corresponds to only 2% of the reference detector area (0.785 mm2). This confirms that the SPFS produces significantly higher output amplitudes despite the small sensing area. The acoustic sensitivity of 417–426 mV/MPa is significantly higher than those of other optical detectors designed for high-amplitude pressure measurement; for example, 6 mV/MPa for a commercially-available fiber-optic hydrophone [In terms of peak signal amplitudes in drophone  and 65.4drophone .In order to determine noise levels of SPFS and PVDF hydrophone, we chose a 1.5-μs period shown in the bottom of We also determined the NEP at 7 MHz. The rms noise voltage for the photoreceiver bandwidth (1–125 MHz) was first measured without any acoustic input. This value was then multiplied by a factor of 3 to determine the peak noise voltage that can be used as a realistic indication for broadband signal measurement . By divi2, see For the above noise analysis using the 7-MHz concave source, we avoided choosing the crosstalk signal shown between −2 and −1 μs range in The SPFS is a robust ultrasound sensor because there is no additional coating on the polished glass surface. Device robustness was tested by using high input pressure amplitudes with an order of MPa. The 7-MHz piezoelectric transducer was used as a calibrated source. In 2, respectively. For each length, the measurement was repeated multiple times by laterally moving the SPF . The SPFS response was further investigated by varying the detector length of SPFS. The SPFS output should increase with the sensing length and thus volume. The exposed SPF length was truncated on both ends by covering them with two glass slides, only leaving a central part of the SPFS exposed and in contact with water. Because of acoustic impedance mismatch and attenuation, the glass plates effectively excluded acoustic transmission (measured reduction > 20 dB). The exposed length (d) at the center was set to 0.1, 0.5, 0.8, 1.2, 1.5 and 2 mm, corresponding to the area of 0.0008, 0.004, 0.0064, 0.0096, 0.012 and 0.016 mm2 with no additional coatings, it exhibits the high sensitivity of 417 and 426 mV/MPa at 3- and 7-MHz frequencies together with the NEP of 6.6 kPa (at 7 MHz) over 1–50 MHz bandwidth. Furthermore, as the sensor is operated with the bare polished surface, it allows for mechanical robustness that is suitable for reliable measurement of high-pressure amplitudes of the order of MPa. We expect that our structure and operation concept can not only be extended to the array platforms using multiple SPFs but can also be utilized to develop high-frequency and high-resolution ultrasound imaging systems, fiber integrated ultrasound endoscopes, and all-optical ultrasound transducers. We have demonstrated a fiber-optic ultrasound sensor using the SPF structure, featuring a broad frequency bandwidth over dc–46 MHz and a wide amplitude dynamic range of several kPa–4.8 MPa, confirmed by using photoacoustic transmitters and piezoelectric transducers as acoustic sources. The SPF uses a microscale-roughened structure for evanescent-field interaction with water that is acoustically modulated. Although the SPFS has a small detection area of only 0.016 mm


Serum and tissue miR-21 expression in patients with breast cancer (BC) is a useful biomarker for cancer diagnosis, progression, and treatment. Matrix metalloproteinase-1 (MMP-1) is also important in breast cancer carcinogenesis. However, miR-21 and MMP-1/CD63 in urine exosomes in these patients have not been examined. Urine samples were collected from patients with BC and 26 healthy females. Urinary exosomes were isolated and confirmed by western blotting with anti-CD63 antibody and electron microscopy observation. MiR-21 and MMP-1/CD63 expression was examined by quantitative RT-PCR and western blotting, respectively. Patients with very early stage breast cancer were evaluated. MiR-21 expression in the patients was 0.26 [95% CI: 0.20–0.78], which was significant lower than in the 26 controls . MMP-1/CD63 expression in patients was significantly higher than in controls . Sensitivity and specificity were 0.708 and 0.783 for miR-21 and 0.792 and 0.840 for MMP-1/CD63, respectively. Sensitivity and specificity of combined expression were 95% and 79%, respectively. The sensitivity of MMP-1/CD63 expression in urinary exosomes was better than that of miR-21 expression. Thus, miR-21 and MMP/CD63 may be useful markers for BC screening. In Japan, however, the prevalence of BC has gradually increased, with the highest incidence rate and the third leading cause of mortality among all cancers2. Early detection of BC is associated with a good prognosis, and thus BC screening has been widely promoted for the past 30 years in Japan. However, mammography and/or echography is time-consuming and costly, as well as inconvenient.Breast cancer (BC) is prevalent worldwide. In the United States, the incidence of BC was high in the 1990s, but has somewhat decreased5. However, the reliability of these tests was low. Screening methods for BC that are simple, reliable, easy to access, and inexpensive are needed. Molecular biology studies showed that exosomes secreted from cancer cells can migrate to distant organs to form niches, leading to metastasis7. Exosomes contain both microRNAs (miRs) and oncogenes7. The expression levels of miR-21 in the tissue and serum derived from patients with BC have been reported as useful biomarkers for diagnosis, cancer progression, and treatment selection16. MiR-21 inhibits the function of several tumour suppressor genes18. The expression of miR-21 in urinary exosomes of patients with BC has not been reported because they had not confirmed their samples by CD63 antibody nor by the electron microscopy observation16.We previously reported urinary cancer screening tests for measuring urinary 3-hdroxyproline23 and development of hepatocellular carcinoma26. MMP-1 is a key proteinase in matrix metabolism27. MMP-1 not only attacks collagen molecules at the three quarters from N-terminals27 but also is involved in cancer development including BC29.We also previously investigated the mechanism of matrix metalloproteinase-1 (MMP-1) in the formation of liver cirrhosisThis study was performed to determine the expression levels of both miR-21 and MMP-1/CD63 in urinary exosomes and evaluate these biomarkers for screening of early stage BC.in situ.The clinical characteristics and histopathological findings of the 22 patients with BC are shown in Table Urinary exosomes isolated with a Miltenyi Biotec isolation kit  were confirmed to be exosomes by western blotting with anti-CD63 antibody Fig.  and the The relative expression levels of miR-21 in 22 patients with BC based on the expression levels of healthy controls are shown in Table The expression levels of MMP-1/CD63 in 22 patients are shown in Table The sensitivity of miR-21 in BC patients was 0.708, and the specificity of miR-21 was 0.792 in primary screening for BC against the 26 healthy controls, as shown in Fig. For miR-21, values less than the cut-off level value 0.413) were considered as positive in screening by receiver operating characteristics (ROC) analysis. For MMP-1/CD63, values higher than the cut-off level value (0.916) were considered as positive in screening. Table  were conSeven cases provided urine samples at 20–136 weeks after surgical treatments Table . Post-opThe expression levels of MMP-1/CD63 were recovered to a normal range in four of seven patients after surgical intervention, although postoperative MMP-1/CD63 expression was not significant but showed a decreasing tendency Table . DramatiCombined expression analysis revealed normal ranges in four of seven cases Table . Case NoAccording to recent clinical classification for treatment selection, Luminal A, Luminal B, Her2, and triple-negative were analysed to detect oestrogen receptor (ER), progesterone receptor (PR), and Her2 levels . The correlations between both expression levels and age (less than 49 years old vs more than 50 years old), body weight, creatinine, carcinoembryonic antigen (CEA), CA15-3, Ki67, alcohol drinking habit, and smoking were analysed. A significant correlation was detected between miR-21 and body weight , Ki67  and creatinine , while MMP-1/CD63 showed no correlation.Several excellent screening methods for BC are available, including physical examination, mammography, ultrasonography, PET-CT, and MRI. Although these examinations are widely performed, they are costly and time-consuming. We developed a novel primary screening test based on urine collection that is inexpensive and convenient. The present study revealed that the combined expression levels of miR-21 and MMP-1/CD63 in urine exosomes can detect 95% of early BC without metastasis. However, there were some limitations. This was a pilot study performed in a single centre and few patients were available for analysis. Additional patients should be evaluated in further studies.30. When only miR-21 is positive, other cancers 30 and liver fibrosis should be considered31. When only MMP-1 is positive, although lung cancer, prostate carcinoma, hepatocellular carcinoma, and other malignancies are considered28, BC should be suspected, particularly in younger women. The expression mechanism of MMP-1 in BC was recently clarified29. Ma et al.29 reported that miR-361-5p inhibited the proliferation of BC cells by suppressing glycolysis and inhibited BC cell invasion and metastasis by targeting MMP-1. Patients with bone disease, skin disease, and rheumatoid arthritis may also show positive results in this test.Detection of the expression of both miR-21 and MMP-1/CD63 in urine exosomes as a BC screening has not been reported previously. miR-21 and MMP-1 play very different biological roles, and thus their expression levels differed. miR-21 and MMP-1 are not specific to BC16. Erbes, et al.16 noted that their method was equivalent to using exosome isolation, but they did not confirm the exosomes themselves. Their results for the expression of miR-21 were consistent with those determined in this study.For miR-21, the results of the present study were consistent with those of numerous previous reports of plasma or serum miR-21 without exosome isolation, which has been reported as a useful biomarker for the diagnosis, treatment selection, and future prediction of BC32. In the present study, no cases showed higher levels of CEA and/or CA 153 (Supplementary Table The serum levels of CEA and carbohydrate antigen 153 (CA 153) have been reported as circulating biomarkers for BC. They are not useful for detecting early BC and have been used as prognostic markers for monitoring disease progression or recurrence33, although the number of cases in the present study was low. Further studies are necessary to confirm the usefulness of both biomarkers.Although the expression levels of miR-21 and MMP-1 in urine exosomes were not specific for BC, the present study showed good results. Ninety-five percent of BC cases were detected compared to using combined expression of both biomarkers. This percentage does not differ from those of Cohen34. The present study revealed that the sensitivity for Luminal B, Her2, and TN cases was 100%, although Luminal A showed a value of 75%. A clear correlation between the percentages of ER-positive cells, PR-positive cells, and HER2-positive cells and the expression levels of miR-21 and MMP-1 were observed. Blenkiron et al.35 showed that elevated miR-21 was associated with ER-positive tumours, and Huang et al.36 reported that miR-21 was up-regulated via the MAP (ERK1/2) pathway upon stimulation of HER2 signalling in BC cells. A correlation between miR-21 and PR has not been reported37. These issues must be resolved to improve the selection of BC treatment, and measuring the expression of miR-21 in urinary exosomes is a convenient approach.We could not conclude whether miR-21 from urine exosomes is equal to plasma or serum exosomal miR-21. MiR-21 from urine exosomes may be useful for selecting treatments after pathological diagnosis can reveal the metastasis of axillary lymph node and/or sentinel lymph node. Thus, decision making for the selection of radiation and/or chemotherapy requires miR-21 urine exosomes38. Melo et al.38 showed that exosomes from BC cells transfer miRs to normal cells and stimulate them to become cancerous. Higher miR-21 expression may be related to suppression of programmed cell death 417, tumour suppressor gene TPM118, and mapin (a tumour suppressing serpin protein)39. Liu et al. reported that miR-21 induced angiogenesis through AKT and ERK activation and HIF1α expression40. Moreover, Fabbri et al. showed that tumour-secreted miR-21 binds to TLR8 to trigger a Toll-like receptor-mediated pro-metastatic inflammatory response that ultimately may lead to tumour growth and metastasis41. We observed one patient with multiple bone marrow metastasis after surgical treatment who showed very low levels of miR-21 (0.01) before operation, whereas high levels (17.78) were detected after the occurrence of metastasis , the Kitasato University Medical Center , and the International University of Health and Welfare Hospital  between July 1, 2016 and November 30, 2018. Prior to implementation, the study was approved by the ethics committee of the three institutions . All patients provided informed consent by formal letter for this study. Twenty-two patients with BC were diagnosed by mammography, echography, MRI, and subsequent needle biopsy before surgery, and the final pathological diagnosis was delivered by surgical resection at Sanno Hospital  classification system46  for 10 min. They were stained with 2% uranyl acetate for 2 min. Observation was performed with a transmission electron microscope .All samples were transferred to Kitasato University Medical Center at −80 °C and urinary exosomes were isolated to analyse the expression of miR-21 by RT-PCR and the expression of MMP-1/CD63 by western blotting. Urinary exosomes were isolated with an Exosome Isolation Kit (Miltenyi Biotec) and were confirmed to be exosomes by western blotting with biotin-conjugated anti-CD63 antibody . The isolated exosomes were also identified by electron microscopyg for 15 min at 4 °C and passed through 0.22-μm nylons filter. The RNA was extracted from isolated exosomes using a Total Exosome RNA & Protein Isolation Kit , and cDNA was synthesized from the acquired microRNA template with a reverse transcription kit . We performed miRNA extraction after quantifying the amount of urine exosomes using a PS Capture Exosome ELISA Kit . Furthermore, the miRNA amount between numerous samples was corrected by performing a reverse transcription reaction using 5 ng of the collected miRNA for expression analysis.A volume of 2 mL urine was used from all samples collected from patients with BC and healthy controls for exosome isolation. Before exosome isolation, the thawed urine was centrifuged at 3,000 × ®  for miR extracted from urinary exosomes. An miR-21 assay (Applied Biosystems) was performed for miR expression assays according to the manufacturer’s instructions. Assays were performed in duplicate tubes for each sample.Quantitative RT-PCR was carried out in a Step One PlusΔCT. Similarly, the value was compared to preoperative and postoperative expression in 7 patients with BC.Based on the mean number of miR-21 copies in the 26 healthy controls, the copy number of miR-21 in patients with BC was calculated as the relative expression grade. ΔCT was determined by subtracting the mean CT value of miR-21 in healthy controls from the individual CT value of miR-21 in patients with BC. Finally, the copy number of miR-21 was compared as the value of 247. Similarly, preoperative and postoperative expression was compared in 7 patients with BC, which revealed identical results. To validate reproducibility, all experiments were performed twice, and the average or median value was confirmed.The expression levels of MMP-1 and CD63 were determined by western blotting. Expression of MMP-1/CD63 was examined using an anti-MMP-1 antibody  and biotin-conjugated anti-CD63 antibody (BioLegend). Their expression were pixeled and the ratio of MMP-1/CD63 in the 22 patients with BC was compared to those in the 26 controls with Image J 1.52a softwaret test. Mann-Whitney U tests were used to evaluate differences between groups: a p value < 0.05 was regarded as statistically significant.All values are expressed as the mean ± standard deviation (SD) or median [95% confidence interval (CI)]. Statistical analysis was performed using PRISM 7 for Mac OS X . Data were tested for normality and equal variance to confirm the appropriateness of parametric tests. Data followed a normal distribution were analysed by Student All procedures performed in this study were in accordance with the ethical standard of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the ethics committees of the three institutions . This article does not contain any studies with animals performed by any of the authors.Measuring the expression of both miR-21 and MMP-1/CD63 in urine exosomes is an effective, reliable, inexpensive, and simple screening test. Further studies are needed to confirm these results in a larger sample size.Supplementary material